WNT Signaling Pathway ChIP PCR Array

The Mouse WNT Signaling Pathway EpiTect ChIP qPCR Array profiles the histone modification status or “histone code” of 84 genes key to WNT-mediated signal transduction. Histone modifications regulate chromatin structure and correlate with the transcriptional activity of associated genes. The WNT family of secreted growth factors regulates the developmental processes of cell fate and polarity, as well as general cell maintenance processes such as homeostasis and cell cycle regulation. There are 19 WNT ligands in humans, which bind to the Frizzled (FZD) family of receptors and the co-receptors LRP5 and LRP6. WNT signaling comprises 3 pathways: the canonical pathway and 2 non-canonical pathways, planar cell polarity (PCP) and a calcium ion-dependent pathway. The well-studied canonical WNT pathway signals through ß-catenin and regulates the cell cycle, cell growth, and proliferation. The PCP pathway regulates cytoskeletal dynamics and cell motility, and the WNT/calcium pathway promotes NFAT transcription, both independently of ß-catenin signaling. Dysregulation of WNT signaling can occur during carcinogenesis and developmental defects, and the underlying mechanisms may include epigenetic regulation. This array contains WNT signaling ligands and receptors as well as other downstream signaling molecules for all 3 pathways. In addition, regulators of WNT signaling are included as well as downstream target genes. Using chromatin immunoprecipitation and this real-time PCR array, research studies can easily and reliably analyze the histone modification patterns associated with a focused gene panel involved in WNT-mediated signal transduction.

EpiTect Chip qPCR Arrays provide a reliable tool for the analysis of a
panel of ChIP-enriched genomic sequences associated with transcription
factors and co-regulators, modified and unmodified histones, and other
DNA-binding proteins. The ChIP PCR Arrays can be used for research on stem
cells, cancer, immunology, stem cells, toxicology, biomarker discovery and
validation.

The ChIP PCR array is a set of optimized real-time PCR primer assays on 96-well
or 384-well plates for pathway or disease focused analysis of in vivo
protein-DNA interactions. The ChIP PCR array performs ChIP DNA analysis with
real-time PCR sensitivity and the multi-genomic loci profiling capability of a
ChIP-on-chip. Simply mix your ChIP DNA samples with the appropriate ready-to-use
PCR master mix, aliquot equal volumes to each well of the same plate, and then
run the real-time PCR cycling program. (Download user manual)

What ChIP PCR Array Offers?

Function or Disease Focused: Arrays represent a panel of
genomic regions relevant to a biological function or disease state.

Easy to Use Data Analysis: Download an easy-to-use Excel-based data
analysis template [here]. Data analysis is based on the ΔΔCt method with
normalization of the specific antibody and control IgG raw data to input
raw data.

Layout and Controls: The PCR Arrays are available in both 96- and
384-well plates and are used to monitor the expression of 84 genes related to a
disease state or pathway plus five housekeeping genes. Controls are also
included on each array for ChIP DNA quality controls and general PCR
performance.
You can easily perform a ChIP PCR Array experiment in your own laboratory, or
send your samples to us and take advantage of our PCR
Array Services.

Table 1. ChIP PCR Arrays Analyze the Enrichment of 84 Genomic Sites with
as Little as One Million Cells. P19 mouse embryonic carcinoma cells were
prepared for ChIP Assay using the EpiTect Chip One-Day Kit and anti-H3K4me3
Antibody Kit. One million cells were used as starting material for each ChIP
Assay. The purified ChIP DNA samples were characterized using Mouse Stem Cell
Transcription Factor ChIP PCR Array with 1/100th of the ChIP DNA as template in
each well. The Real-Time PCR results demonstrate 100 % effective call rates for
the Input Fraction (Ct < 30). The difference of Ct value between the
anti-H3K4me3 antibody and the control IgG fractions indicates the specific
enrichment of the antibody, whereas the high Ct value of the control IgG
fraction indicates the low background of the assay.

ReproducibilityThe complete ChIP PCR Array System demonstrates a high degree of
reproducibility across technical replicates, lots, instruments, and different
handling, insuring reliable detection of differences in genomic DNA enrichment
among biological samples.

Figure 5. Consistent Performance within the Same Plate or across Different
Plates. Sonicated chromatin from HeLa cells (20 µg) was immunoprecipitated
with 2 µg of anti-H3ac antibody or control IgG for 2 hours using the
EpiTect Chip One-Day Kit. The obtained ChIP DNA samples were characterized in
triplicates with EpiTect Chip qPCR primers specific for the active genes (GAPDH,
RPL30, ALDOA), inactive genes (MYOD1, SERPINA), repetitive sequence (SAT2, SATa),
and an ORF-free region (IGX1A) either within the same array plate or among
different array plates in order to evaluate the intra- and inter-plate
consistency. The anti-H3ac antibody enriched genomic DNA at active gene promoter
regions with a high signal-to-noise ratio and a low co-efficiency of variation
(less than 2.02%), irrespective of the type of assay (intra or inter-plate)

Figure 6. Consistent Performance with Various Amount of DNA Samples,
Instruments or Handling Conditions. All experiments were performed in
triplicates. Cells from MCF-7 (1 million per sample) were subjected to ChIP
assay with anti-RNA Polymerase II (Pol 2) antibody followed by qPCR analysis of
the proximal promoter of GAPDH, and an ORF-free region (IGX1A). Researcher A
& B performed the PCR assays either in 96-well plate or 384-well plate
format, on a Stratagene MX 3005 or an ABI 7900 Real-Time PCR instrument
respectively. The same ChIP DNA samples were used which were stored for extended
periods of time as indicated. The results demonstrate high reproducibility of
PCR performance across technical replicates, lots, instruments, and differential
handling.

Figure 7. Uniform Amplification Efficiency and Specific PCR Detection.
96 ChIP-qPCR primers were randomly picked from our genome-wide primer pool and
analyzed for their performance. (A) All assays exhibit an average amplification
efficiency of 99% with a 104.5% confidence interval between 102.5-105.2%, the
uniform high amplification efficiency ensures accurate analysis of multiple
genomic loci simultaneously using ΔΔCt method. (B) Each ChIP-qPCR primer assay
is experimentally validated using dissociation (melt) curve analysis and agarose
gel verification. Each pair of primers on PCR Array produces a single specific
product as indicated by a single Dissociation Curve peak at a melting
temperature (Tm) greater than 75 ºC, and PCR product was further validated
on agarose gel for a single product of the predicted size without secondary
products such as primer dimers

Figure 3. Dynamic Epigenetic Alternations and Gene Expression
Changes during RA-Induced P19 Differentiation. ChIP PCR Arrays and RT² PCR
Arrays were used to monitor the changes in gene expression levels and histone
modification marks (H3Ac, H3K4me3, H3K27me3, and H3K9me3). The promoter region
and expression levels of 84 key stem cell transcription factors were
simultaneously analyzed during RA-induced neurogenesis of P19 cells at various
time points (day 0, 4, and 8). Primer sets for the +1kb region downstream of the
transcription start sites of the 84 genes and 12 control regions were preloaded
on the ChIP PCR Array. Cluster analysis (http://www.sabiosciences.com/chippcrarray_data_analysis.php)
of histone marks and mRNA level changes for the 84 genes were visualized as a
heat map to represent the fold-differences during the RA-induced differentiation
at the specified time points.

Characterize the Pattern of Histone Modifications

EpiTect Chip qPCR Arrays can be used to monitoring differential
histone modifications across a gene.

Figure 4. The Custom EpiTect Chip 30Kb Tiling Array Quickly
Maps Histone Modifications Surrounding the Transcription Start Site (TSS) of
CDKN1A Gene. EpiTect Chip Antibodies against modified histones (H3Ac,
H3K4me2, H3K27me3), or NIS were used to precipitate chromatin from one million
HeLa cells. Each ChIP DNA fraction was analyzed with Custom EpiTect Chip 30Kb
Tiling Array representing 30 one-kb tile intervals across the promoter region of
the CDKN1A gene. The results indicate the enrichment of histone markers for
actively transcribed genes (H3Ac and H3K4me2) but not marks for transcriptional
inactive genes (H3K27me3) in the genomic region surrounding the TSS of CDNK1A.